The present invention relates to enzymatic processes for the resolution of enantiomeric mixtures of compounds useful as intermediates in the preparation of taxanes, particularly for the preparati of taxanes bearing a C-13 sidechain containing a heterocyclic or cycloalkyl group.
Taxanes are diterpene compounds which find utility in the pharmaceutical field. For example, taxol analogues containing heterocyclic or cycloalkyl groups on the C-13 sidechain find utility as anticancer agents. Such taxol analogues may be prepared through semi-synthetic routes, particularly by the coupling of xcex2-lactam or open chain intermediates to the taxane core to form a sidechain at C-13. As the stereochemistry of these analogues may affect their pharmaceutical activity, methods allowing efficient stereospecific preparation of the intermediate xcex2-lactam and open chain compounds, as well as the final taxane products, are sought in the art.
The present invention provides efficient methods for the resolution of enantiomeric mixtures, preferably racemic mixtures, of compounds useful as intermediates in the preparation of taxanes bearing a C-13 sidechain containing a heterocyclic or cycloalkyl group, and thus for the stereospecific preparation of these compounds.
Specifically, the present invention provides a method for the resolution of a mixture I comprising the enantiomers Ia and Ib, where R1 is in the cis position relative to R2 in both Ia and Ib, or where R1, is in the trans position relative to R2 in both Ia and Ib: 
where
R1 is hydroxyl; halo; or xe2x80x94Oxe2x80x94C(O)xe2x80x94R4, where R4 is alkyl, alkenyl, alkynyl, aryl, cycloalkyl, cycloalkenyl or heterocyclo;
R2 is heterocyclo or cycloalkyl; and
R3 is hydrogen; R4; xe2x80x94C(O)xe2x80x94OR ; or xe2x80x94C(O)xe2x80x94R4, where R4 is independently selected from those groups recited for R4 above;
comprising the step of contacting said mixture I with an enzyme or microorganism capable of catalyzing the stereoselective conversion of one of said compounds Ia or Ib to a non-enantiomeric form, and effecting said conversion.
The present invention also provides a process for the resolution of a mixture IV comprising the enantiomers IVa an IVb:
R2xe2x80x94Taxe2x80x94C(O)xe2x80x94OR6xe2x80x83xe2x80x83(IVA)
and
R2xe2x80x94Tbxe2x80x94C(O)xe2x80x94OR6xe2x80x83xe2x80x83(IVb)

where
R1 is in the erythro position relative to the group W in both IVa and IVb, or where R1 is in the threo position relative to the group W in both IVa and IVb;
W is xe2x80x94NHR or xe2x80x94N3;
R1 is hydroxyl; halo; or xe2x80x94Oxe2x80x94C(O)xe2x80x94R4, where R4 is alkyl, alkenyl, alkynyl, aryl, cycloalkyl, cycloalkenyl or heterocyclo;
R2 is heterocyclo or cycloalkyl;
R3 is hydrogen; R4; xe2x80x94C(O)xe2x80x94OR4; or xe2x80x94C(O)xe2x80x94R4, where R4 is independently selected from those groups recited for R4 above; and
R6 is hydrogen; or R4, where R4 is independently selected from those groups recited for R4 above;
comprising the step of contacting said mixture IV with an enzyme or microorganism capable of catalyzing the stereoselective conversion of one of said compounds IVa or IVb to a non-enantiomeric form, and effecting said conversion.
Exemplary embodiments for the aforementioned stereoselective conversions include stereoselective hydrolysis, stereoselective esterification, stereoselective transesterification and stereoselective dehalogenation, particularly stereoselective hydrolysis or esterification.
Groups, such as hydroxyl groups, on the compounds of formulae I or IV may optionally be protected for use in the resolution methods of the present invention; such groups may optionally be subsequently deprotected.
The methods of the present invention are described further as follows.
Cis Enantiomers
The following pair of cis enantiomers may be separated by the enzymatic methods of the instant invention: 
that is, enantiomers Ia and Ib where R1 is in the cis position relative to R2 in both Ia and Ib.
It is preferred to resolve a mixture of cis enantiomers as described above according to the methods of the instant invention.
Trans Enantiomers
The following pair of trans enantiomers may be separated by the enzymatic methods of the instant invention: 
that is, enantiomers Ia and Ib where R1 is in the trans position relative to R2 in both Ia and Ib.
Erythro Enantiomers
The following pairs of erythro enantiomers may be separated by the enzymatic methods of the instant invention: 
that is, enantiomers IVa and IVb where R1 is in the erythro position relative to the group W in both IVa and IVb.
Threo Enantiomers
The following pairs of threo enantiomers may be separated by the enzymatic methods of the instant invention: 
that is, enantiomers IVa and IVb where R1 is in the threo position relative to the group W in both IVa and IVb.
Mixture I, comprising an enantiomeric mixture of xcex2-lactams Ia and Ib, is preferably resolved by stereoselective hydrolysis, esterification or dehalogenation. A particularly preferred method for the resolution of a mixture I comprising the enantiomers Ia(1) and Ib(1): 
to form a mixture II comprising the compounds IIa(1) and IIb(1): 
where
R2 is heterocyclo or cycloalkyl; and
R3 is hydrogen; R4; xe2x80x94C(O)xe2x80x94OR ; or xe2x80x94C(O)xe2x80x94R4, where R4 is alkyl, alkenyl, alkynyl, aryl, cycloalkyl, cycloalkenyl or heterocyclo;
comprises one of the following steps (i), (ii), or (iii):
(i) where
R1 is xe2x80x94Oxe2x80x94C(O)xe2x80x94R4, where R4 is independently selected from those groups recited for R4 above; and one of R1a or R1b is the same as R1 and the other of R1a or R1b is hydroxyl;
the step of contacting said mixture I, in the presence of water and/or an organic alcohol, with an enzyme or microorganism capable of catalyzing the stereoselective hydrolysis of mixture I to provide said mixture II; or
(ii) where
R1 is hydroxyl; and
one of R1a or R1b is hydroxyl and the other of R1a or R1b is R4xe2x80x94C(O)xe2x80x94Oxe2x80x94, where R4 is independently selected from those groups recited for R4 above;
the step of contacting said mixture I, in the presence of a compound III:
R4xe2x80x94C(O)xe2x80x94Lxe2x80x83xe2x80x83(III)
where R4 is as defined above for R1a or R1b and L is a leaving group, with an enzyme or microorganism capable of catalyzing the stereoselective esterification of mixture I to provide said mixture II; or
(iii) where
R1 is a halogen atom; and
one of R1a or R1b is halogen and the other of R1a or R1b is hydroxyl;
the step of contacting said mixture I, in the presence of a hydroxide ion donor, with an enzyme or microorganism capable of catalyzing the stereoselective dehalogenation of mixture I to provide said mixture II.
The above methods may be employed in the resolution of other enantiomeric mixtures of the instant invention, although resolution of the above cis enantiomers Ia(1) and Ib(1) is preferred.
Mixture IV is preferably resolved by stereoselective hydrolysis, esterification, dehalogenation or transesterification. A particularly preferred method for the resolution of a mixture IV comprising the enantiamers IVa(l) and IVb(1): 
to form a mixture V comprising compounds Va(1) and Vb(1); 
where
R2 is heterocyclo or cycloalkyl;
R3 is hydrogen; R4; xe2x80x94C(O)xe2x80x94OR4; or xe2x80x94C(O)xe2x80x94R4, where R4 is alkyl, alkenyl, alkynyl, aryl, cycloalkyl, cycloalkenyl or heterocyclo; and
R6 is hydrogen; or R4, where R4 is independently selected from those groups recited for R4 above;
comprises one of the following steps (i), (ii), or (iii):
(i) where
R1 is xe2x80x94Oxe2x80x94C(O)xe2x80x94R4, where R4 is independently selected from those groups recited for R4 above; and one of R1a or R1b is the same as R1 and the other of R1a or R1b is hydroxyl;
the step of contacting said mixture IV, in the presence of water and/or an organic alcohol, with an enzyme or microorganism capable of catalyzing the stereoselective hydrolysis of mixture IV to provide said mixture V; or
(ii) where
R1 is hydroxyl; and
one of R1a or R1b is hydroxyl and the other of R1a or R1b is R4xe2x80x94C(O)xe2x80x94Oxe2x80x94, where R4 is independently selected from those groups recited for R4 above;
the step of contacting said mixture IV, in the presence of a compound III:
R4xe2x80x94C(O)xe2x80x94Lxe2x80x83xe2x80x83(III)
where R4 is as defined above for R1a or R1b and L is a leaving group, with an enzyme or microorganism capable of catalyzing the stereoselective esterification of mixture IV to provide said mixture V; or
(iii) where
R1 is a halogen atom; and
one of R1a or R1b is halogen and the other of R1a or R1b is hydroxyl;
the step of contacting said mixture IV, in the presence of a hydroxide ion donor, with an enzyme or microorganism capable of catalyzing the stereoselective dehalogenation of mixture IV to provide said mixture V.
A further particularly preferred method for the resolution of a mixture IV comprising the enantiomers IVa(1) and IVb(1): 
to form a mixture VI comprising compounds VIa(1) and VIb(1): 
where
R1 is hydroxyl; halo; or xe2x80x94Oxe2x80x94C(O)xe2x80x94R4, where R4 is alkyl, alkenyl, alkynyl, aryl, cycloalkyl, cyloalkenyl or heterocyclo;
R2 is heterocyclo or cycloalkyl; and
R3 is hydrogen; R4; xe2x80x94C(O)xe2x80x94OR ; or xe2x80x94C(O)xe2x80x94R4, where R4 is independently selected from those groups recited for R4 above;
comprises one of the following steps (i), (ii), or (iii):
(i) where
R6 is hydrogen; and one of R6a or R6b is hydrogen and the other of R6a or R6a is R4, where R4 is independently selected from those groups recited for R4 above;
the step of contacting said mixture IV, in the presence of an organic alcohol of the formula VII:
R4xe2x80x94OHxe2x80x83xe2x80x83(VII)
where R4 is as defined above for R6a or R6b with an enzyme or microorganism capable of catalyzing the stereoselective esterification of mixture IV to provide said mixture VI; or
(ii) where
R6 is R4, where R4 is independently selected from those groups recited for R4 above; and
one of R6a or R6b is the same as R6 and the other of R6a or R6b is hydrogen;
the step of contacting said mixture IV, in the presence of water, with an enzyme or microorganism capable of catalyzing the stereoselective hydrolysis of mixture IV to provide said mixture VI; or
(iii) where
R6 is R4, where R4 is independently selected from those groups recited for R4 above; and
one of R6a or R6b is the same as R6 and the other of R6a or R6b is R7, where R7 is alkyl, alkenyl, alkynyl, aryl, cycloalkyl, cycloalkenyl or heterocyclo, except that R7 is not the same as R6;
the step of contacting said mixture IV, in the presence of an organic alcohol of the formula VIII:
R7xe2x80x94OHxe2x80x83xe2x80x83(VIII)
where R7 is as defined above, with an enzyme or microorganism capable of catalyzing the stereoselective transesterification of mixture IV to provide said mixture VI.
The above methods may be employed in the resolution of other enantiomeric mixtures of the instant invention, although resolution of the above enantiomers IVa(1) and IVb(1) is preferred.
The compound pairs so prepared, such as IIa(1) and IIb(1), are non-enantiomeric and may subsequently be separated to yield optically active, preferably optically pure, compounds. An optical purity greater than 99%, particularly 99.5%, is preferred.
The instant invention also provides a compound of the mixture I or IV substantially free of other isomers, which compound may be prepared by the methods of the invention.
The term xe2x80x9cstereoselective conversionxe2x80x9d, as used herein, refers to the preferential reaction of one enantiomer relative to another, that is, asymmetric, enantioselective, reaction. Likewise, the terms xe2x80x9cstereoselective hydrolysisxe2x80x9d, xe2x80x9cstereoselective esterificationxe2x80x9d, stereoselective dehalogenationxe2x80x9d and xe2x80x9cstereoselective transesterificationxe2x80x9d refer to the preferential hydrolysis, esterification, dehalogenation and transesterification, respectively, of one enantiomer relative to another.
The term xe2x80x9cmixturexe2x80x9d, as said term is used herein in relation to enantiomeric compounds, denotes mixtures having equal (racemic) or non-equal amounts of enantiomers.
The term xe2x80x9cresolutionxe2x80x9d as used herein denotes partial, as well as, preferably, complete resolution.
The term xe2x80x9cnon-enantiomeric formxe2x80x9d as used herein denotes the structure of a compound, originally one of an enantiomeric pair, in which at least one group has been modified so that said compound is no longer the mirror image of the other compound of the original enantiomeric pair.
The terms xe2x80x9cenzymatic processxe2x80x9d or xe2x80x9cenzymatic methodxe2x80x9d as used herein denote a process or method of the present invention employing an enzyme or microorganism.
The terms xe2x80x9calkylxe2x80x9d, xe2x80x9calkanxe2x80x9d or xe2x80x9calkxe2x80x9d as employed herein alone or as part of another group denote both straight and branched chain, optionally substituted hydrocarbons groups containing 1 to 15 carbons in the normal chain, preferably 1 to 6 carbons, such as methyl, ethyl, propyl, isopropyl, butyl, t-butyl, isobutyl, pentyl, hexyl, isohexyl, heptyl, 4,4-dimethylpentyl, octyl, 2,2,4-trimethyl-pentyl, nonyl, decyl, undecyl, dodecyl, the various branched chain isomers thereof, and the like. Exemplary substituents include one or more groups selected from the following: halo (especially chloro), trihalomethyl, alkoxy (for example, where two alkoxy substituents form an acetal), aryl such as unsubstituted aryl, alkyl-aryl or haloaryl, cycloalkyl such as unsubstituted cycloalkyl or alkyl-cycloalkyl, hydroxy or protected hydroxy, carboxyl, alkyloxycarbonyl, alkylamino, alkylcarbonylamino, amino, arylcarbonylamino, nitro, cyano, thiol or alkylthio.
The term xe2x80x9calkenylxe2x80x9d as employed herein alone or as part of another group denotes such optionally substituted groups as described above for alkyl, further containing at least one carbon to carbon double bond. Exemplary substituents include one or more alkyl groups as described above, and/or one or more groups described above as alkyl substituents.
The term xe2x80x9calkynylxe2x80x9d as employed herein alone or as part of another group denotes such optionally substituted groups described above for alkyl, further containing at least one carbon to carbon triple bond. Exemplary substituents include one or more alkyl groups as described above, and/or one or more groups described above as alkyl substituents.
The term xe2x80x9ccycloalkylxe2x80x9d as employed herein alone or as part of another group denotes optionally substituted saturated cyclic hydrocarbon groups containing one to three rings and 3 to 12 ring carbons, preferably 3 to 8 ring carbons, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclodecyl, cyclododecyl, and adamantyl. Exemplary substituents include one or more alkyl groups as described above, and/or one or more groups described above as alkyl substituents.
The term xe2x80x9ccycloalkenylxe2x80x9d as employed herein alone or as part of another group denotes such optionally substituted groups as described above for cycloalkyl, further containing at least one carbon to carbon double bond in the ring system. Exemplary substituents include one or more alkyl groups as described above, and/or one or more groups described above as alkyl substituents.
The terms xe2x80x9carylxe2x80x9d or xe2x80x9carxe2x80x9d as employed herein alone or as part of another group denote optionally substituted homocyclic aromatic groups, preferably monocyclic or bicyclic groups containing from 6 to 12 carbons in the ring portion, such as phenyl, biphenyl, naphthyl, substituted phenyl, substituted biphenyl or substituted naphthyl. Exemplary substituents (preferably three or fewer) include one or more of the following groups: alkyl such as unsubstituted alkyl, haloalkyl, or cycloalkyl-alkyl, halogen, alkoxy such as unsubstituted alkoxy or haloalkoxy, hydroxy, aryloxy such as phenoxy, R4-carbonyloxy, where R4 is as defined above, allyl, cycloalkyl, alkylamino, dialkylamino, amido such as alkylcarbonylamino or arylcarbonylamino, amino, nitro, cyano, alkenyl, thiol, R4-carbonyl, where R4 is as defined above, or methylenedioxy where the methylene group may be substituted by 1 or 2 lower alkyl groups, 1 or 2 arylalkenyl groups, and/or 1 or 2 alkylthio groups. Particularly preferred aryl groups are phenyl and substituted phenyl, especially phenyl substituted by one or more hydroxyl, alkyl and/or alkoxy groups.
The terms xe2x80x9chalogenxe2x80x9d or xe2x80x9chaloxe2x80x9d as used herein alone or as part of another group refer to chlorine, bromine, fluorine, and iodine.
The terms xe2x80x9cheterocycloxe2x80x9d or xe2x80x9cheterocyclicxe2x80x9d as used herein alone or as part of another group denote optionally substituted fully saturated or unsaturated, monocyclic or bicyclic, aromatic or nonaromatic hydrocarbon groups having at least one heteroatom in at least one ring, and preferably 5 or 6 atoms in each ring. The heterocyclo group preferably has 1 or 2 oxygen atoms, 1 or 2 sulfur atoms, and/or 1 to 4 nitrogen atoms in the ring, and may be bonded to the remainder of the molecule through a carbon or heteroatom. Exemplary substituents include one or more of the following groups: halogen, alkoxy, hydroxy, aryl such as phenyl or halophenyl, alkanoyloxy, arylcarbonyloxy such as benzoyloxy, alkyl such as aralkyl, alkylamino, alkanoylamino, arylcarbonylamino, amino, nitro, cyano, and thiol. Exemplary heterocyclo groups include thienyl, furyl, pyrrolyl, pyridyl, imidazolyl, pyrrolidinyl, piperidinyl, azepinyl, indolyl, isoindolyl, quinolinyl, isoquinolinyl, benzothiazolyl, benzoxazolyl, benzimidazolyl, benzoxadiazolyl, and benzofurazanyl.
The term xe2x80x9chydroxyl protecting groupxe2x80x9d as used herein denotes a group capable of protecting a free hydroxyl group (xe2x80x9cprotected hydroxylxe2x80x9d) which, subsequent to the reaction for which protection is employed, may be removed without disturbing the remainder of the molecule. A variety of protecting groups for the hydroxyl group and the synthesis thereof may be found in xe2x80x9cProtective Groups in organic Synthesisxe2x80x9d by T. W. Greene, John Wiley and Sons, 1981, or Fiser and Fiser. Exemplary hydroxyl protecting groups include methoxymethyl, 1-ethoxyethyl, benzyloxymethyl, (xcex2-trimethylsilylethoxy)methyl, tetrahydropyranyl, 2,2,2-trichloroethoxycarbonyl, t-butyl(diphenyl)silyl, trialkylsilyl, trichloromethoxycarbonyl and 2,2,2-trichloroethoxymethyl.
The starting materials for the present resolution methods may be obtained as described in the Examples herein, or by methods analogous to those described in U.S. patent application Ser. No. 07/822,015, filed Jan. 15, 1992.
The starting mixtures I or IV may contain, for example, the diastereomers of the compounds Ia and Ib or IVa and IVb, although it is preferred that such compounds are separated prior to conducting the enzymatic resolution methods of the present invention.
Cis compounds of the formula I have a stereoisomeric configuration which is preferred in compounds employed as intermediates in the preparation of C-13 sidechain-bearing taxanes. Compounds of the mixtures I and II having the same absolute stereoconfiguration corresponding to that of a compound Ia where R1 is acetyloxy, R2 is furyl and R3 is hydrogen in the 3R,4S configuration are particularly preferred.
Erythro compounds of the formula IV have a stereoisomeric configuration which is preferred in compounds employed as intermediates in the preparation of C-13 sidechain-bearing taxanes. Compounds of the mixtures IV, V and VI having the same absolute stereoconfiguration corresponding to that of a compound IVa(1) where R1 is hydroxyl, R2 is furyl, W is xe2x80x94NHR3 and R3 is hydrogen, and R6 is hydrogen in the 2R,3S configuration are preferred. In mixture IV, 
is preferred.
Resolution of xcex2-lactams of the formula I is preferred.
In the compounds of the present invention, R1 is preferably alkanoyloxy, such as unsubstituted alkanoyloxy (e.g., acetyloxy), or hydroxy; R2 is preferably furyl or thienyl; and R3 is preferably hydrogen, phenyl, substituted phenyl, phenylcarbonyl, substituted phenylcarbonyl, alkylcarbonyl, alkenylcarbonyl or alkoxycarbonyl such as t-butoxycarbonyl. R6 is preferably hydrogen or a C1-6 alkyl such as methyl.
The enzyme or microorganism employed in the methods of the present invention may be any enzyme or microorganism having the ability to catalyze the stereoseleccive conversions as described herein. Various enzymes, such as esterases, lipases and proteases, regardless of origin or purity, are suitable for use in the present invention. The enzyme may, for example, be in the form of animal or plant enzymes or mixtures thereof, cells of microorganisms, crushed cells, extracts of cells, or of synthetic origin.
With respect to the use of microorganisms, the methods of the present invention may be carried out using any microbial cellular material having the ability to catalyze the stereoselective conversions as described herein. The cells may be used in the form of intact wet cells or dried cells such lyophilized, spray-dried or heat-dried cells. Cells may also be used in the form of treated cell material such as ruptured cells or cell extract.
The enzyme or microbial materials may be employed in the free state or immobilized,on a support (for example, a polymeric resin) such as by physical adsorption or entrapment.
Exemplary genera of microorganisms suitable as sources of catalyzing enzymes include Mucor, Escherichia, Staphylococcus, Agrobacterium, Acinetobacter, Rhizopus, Aspergillus, Nocardia, Streptomyces, Trichoderma, Candida, Rhodotorula, Torulopsis, Proteus, Bacillus, Alcaligenes, Pseudomonas, Rhodococcus, Brevibacterium, Geotrichum, Enterobacter, Chromobacterium, Arthrobacter, Microbacterium, Mycobacterium, Saccharomyces, Penicillium, Methanobacterium, Botrytis, Chaetomium, Ophiobolus, Cladosporium and the like. The use of genetically engineered host cells is also contemplated.
Specific microorganisms suitable for use in the present processes include Chromobacterium viscosum, Pseudomonas aeuriginosa such as ATCC 25619, Pseudomonas fluorescens, Pseudomonas putida such as ATCC 31303, Pseudomonas ovalis, Escherichia coli, Staphylococcus aureus, Alcaligenes faecalis, Streptomyces griseus, Pseudomonas cepacia, Candida rugosa such as ATCC 14830, Geotrichum candidum such as ATCC 32345, Streptomyces clavuligerus, Nocardia erthropolis, Nocardia asteraides, Mycobacterium phlei, Agrobacterium radiobacter, Aspergillus niger, Rhizopus oryzae and the like. Two or more, as well as a single, species of microorganism may be employed when carrying out the instant processes.
The term xe2x80x9cATCCxe2x80x9d as used herein refers to the accession number of the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Md. 20852, the depository for the organism referred to.
The resolution methods of the instant invention may be carried out subsequent to the growth of the microorganism(s) employed, or concurrently therewith that is, in the latter case, by in situ fermentation and resolution. The growth of microorganisms may be achieved by one of ordinary skill in the art, for example, by the use of an appropriate medium containing nutrients such as carbon and nitrogen sources and trace elements.
Exemplary, commercially available enzymes suitable for use in the present invention include lipases such as Amano PS-30 (Pseudomonas cepacia), Amano GC-20 (Geotrichum candidum), Amano APF (Aspergillus niger), Amano AK (Pseudomonas sp.), Pseudomonas fluorescens lipase (Biocatalyst Ltd.), Amano Lipase P-30 (Pseudomonas sp.), Amano P (Pseudomonas fluorescens), Amano AY-30 (Candida cylindracea), Amano N (Rhizopus niveus), Amano R (Penicillium sp.), Amano FAP (Rhizopus oryzae), Amano AP-12 (Aspergillus niger), Amano MAP (Mucor meihei), Amano GC-4 (Geotrichum candidum), Sigma L-0382 and L-3126 (porcine pancrease), Lipase OF (Sepracor), Esterase 30,000 (Gist-Brocarde), KID Lipase (Gist-Brocarde), Lipase R (Rhizopus sp., Amano), Sigma L-3001 (Wheat germ), Sigma L-1754 (Candida cylindracea), Sigma L-0763 (Chromobacterium viscosum) and Amano K-30 (Aspergillus niger). Additionally, exemplary enzymes derived from animal tissue include esterase from pig liver, xcex1-chymotrypsin and pancreatin from pancreas such as Porcine Pancreatic Lipase (Sigma). Two or more, as well as a single, enzyme may be employed when carrying out the instant processes.
The preferred embodiments of the instant invention are described further in the following Reaction Schemes. While, for clarity, these Reaction Schemes illustrate the resolution of certain cis enantiomeric mixtures, it is understood that the embodiments as described apply to the resolution of the other enantiomeric mixtures of the present invention as well. 
Mixtures I and IV may be stereoselectively esterified as illustrated in the above Reaction Scheme I, and mixture IV may be stereoselectively transesterified as illustrated in the above Reaction Scheme II.
(A) Acylation
Mixture I may be selectively esterified to form mixture II, and mixture IV may be selectively esterified to form mixture V by use of an acylating agent of the formula III:
R4xe2x80x94C(O)xe2x80x94Lxe2x80x83xe2x80x83(III)
In formula III, R4 may be an alkyl, alkenyl, alkynyl, aryl, cycloalkyl, cycloalkenyl or heterocyclo group. Preferred R4 groups in formula III are alkyl groups such as C1-6 alkyl groups, especially methyl. L is a leaving group which may be displaced to form an ester group. Exemplary L groups include halogen atoms, hydroxyl, alkoxy, or alkenyloxy groups. Preferred L groups are alkenyloxy groups, most preferably C1-6 alkenyloxy groups such as CH2xe2x95x90CHxe2x80x94Oxe2x80x94 and CH2xe2x95x90C(CH3)xe2x80x94Oxe2x80x94. Any acylation agent of formula III which effects esterification may be employed, with isopropenyl acetate and vinyl acetate being particularly preferred.
(B) Esterification with alcohol
Mixture IV may be selectively esterified to form mixture VI by use of an organic alcohol of the formula VII:
R4xe2x80x94OHxe2x80x83xe2x80x83(VII)
In formula VII, R4 may be an alkyl, alkenyl, alkynyl, aryl, cycloalkyl, cycloalkenyl or heterocyclo group. Alkyl groups, particularly C1-6 alkyl groups, are preferred as R4.
(C) Transesterification with alcohol
Mixture IV may be selectively transesterified to form mixture VI by use of an alcohol of the formula VIII:
R7xe2x80x94OHxe2x80x83xe2x80x83(VIII)
In formula VIII, R7 may be an alkyl, alkenyl, alkynyl, aryl, cycloalkyl, cycloalkenyl or heterocyclo group, except that R7 is not the same as R6. It is preferred that the group R7 be as distinct as possible from the group R6 to facilitate subsequent separation of the compound bearing the group R7xe2x80x94Oxe2x80x94C(O)xe2x80x94 from the compound bearing the group R6xe2x80x94Oxe2x80x94C(O)xe2x80x94. Thus, it is preferred to employ an alcohol of the formula VIII in which the R7 group differs with respect to the group R6 in terms of molecular weight, or otherwise imparts distinctive physical or chemical properties to the transesterified ester.
The esterification (acylation) procedure (A), and the esterification and transesterification procedures (B) and (C), are preferably carried out in an organic solvent. Exemplary solvents suitable for use in these processes include 1,1,2-trichloro-1,2,2-trifluoroethane, toluene, cyclohexane, benzene, hexane, heptane, isooctane, octane, methyl ethyl ketone, methyl isobutyl ketone and the like. Water is preferably added to the reaction mixture in small amounts. When present, the concentration of water in the reaction mixture is preferably from about 0.01% to about 1% based on the weight of solvent, or present in a concentration less than or equal to that where the organic solvent is saturated. Water is most preferably present in an amount of about 0.05% to about 0.5% based on the weight of solvent. The reaction solution preferably contains between about 5 to about 250 mg of the enantiomeric starting compounds per ml of solvent.
To carry out these processes, a compound III, VII or VIII is added to the reaction medium. Preferred molar ratios of the compound III: compounds of mixture I or IV are from about 1:1 to about 4:1; preferred molar ratios of the compound VII: compounds of mixture IV are from about 1:1 to about 4:1; and preferred molar ratios of the compound VIII: compounds of mixture IV are from about 1:1 to about 4:1.
The enzymes or microorganisms employed in these procedures are preferably lipases or esterases or microorganisms capable of producing these enzymes. Enzymes or microorganisms particularly preferred in these processes are Lipase PS-30 from Pseudomonas sp., Lipase P-30 from Pseudomonas sp., Lipase R from Penicillium sp., Lipase OF, Lipase N from Rhizopus niveus, Lipase APF from Aspergillus niger, Lipase GC-20 from Geotrichum candidum, Lipase AK from Pseudomonas sp., Lipase AY-30 from Candida sp., and Pseudomonas fluorescens Lipase.
An enzyme may, for example, be used-in its free state or in immobilized form. A preferred embodiment of the invention is that where an enzyme is adsorbed onto a suitable carrier, e.g., diatomaceous earth (porous Celite Hyflo Supercel), microporous polypropylene (Enka Accurel(copyright) polypropylene powder), or a nonionic polymeric adsorbent such as Amberlite(copyright) XAD-2 (polystyrene) or XAD-7 (polyacrylate) from Rohm and Haas Co. When employed to immobilize an enzyme, a carrier may control the enzyme particle size and prevent aggregation of the enzyme particles when used in an organic solvent. Immobilization can be accomplished, for example, by precipitating an aqueous solution of the enzyme with cold acetone in the presence of the Celite Hyflo Supercel followed by vacuum drying, or in the case of a nonionic polymeric adsorbent, incubating enzyme solutions with adsorbent on a shaker, removing excess solution and drying enzyme-adsorbent resins under vacuum. The enzyme is preferably added to the reaction solution to achieve concentrations ranging from about 5 to about 200 mg of enzyme per ml of solvent. While it is desirable to use the least amount of enzyme possible, the amount of enzyme required will vary depending upon the specific activity of the enzyme used.
These processes may also be carried out using microbial cells containing an enzyme having the ability to catalyze the stereoselective conversions. When using a microorganism to perform the resolution, these procedures are conveniently carried out by adding the cells and the enantiomeric mixture starting material to the desired reaction medium. Cells may be used in the form of intact cells, dried cells such as lyophilized, spray-dried or heat-dried cells, immobilized cells, or cells treated with organic solvents such as acetone or toluene. Cells may also be used in the form of treated cell material such as ruptured cells or cell extract. Cell extracts immobilized on Celite(copyright) or Accurel(copyright) polypropylene as described earlier may also be employed.
Incubation of the reaction medium is preferably at a temperature between about 4 and about 60xc2x0 C. and is most preferably between about 30 to 50xc2x0 C. The reaction time can be appropriately varied depending upon the amount of enzyme used and its specific activity. Reaction times may be reduced by increasing the reaction temperature and/or increasing the amount of enzyme added to the reaction solution. 
As can be seen from Reaction Scheme III above, mixtures I and IV may be stereoselectively hydrolyzed to form mixtures II and V, respectively, by use of water and/or an organic alcohol, and mixture IV may be stereoselectively hydrolyzed to form mixture VI by use of water. The groups R4, forming part of R1, and R6 in the starting enantiomeric compounds are preferably alkyl, most preferably C1-6 alkyl such as methyl.
A compound of the formula IX:
R8xe2x80x94OHxe2x80x83xe2x80x83(IX)
may be employed as the organic alcohol, where R8 is an alkyl, alkenyl, alkynyl, aryl, cycloalkyl, cycloalkenyl or heterocyclo group, and is preferably alkyl such as methyl. Use of the organic alcohol IX may result in the formation of the by-product ester R4xe2x80x94C(O)xe2x80x94OR . Use of water as the hydrolysis agent may result in the formation of the by-product acid R4xe2x80x94C(O)xe2x80x94OH. To maintain a steady pH as these acidic by-products are generated, a base such as an alkali metal hydroxide may be added. When an organic alcohol IX is employed, an amount providing a molar ratio of compound IX: compounds of mixtures I or IV of from about 1:1 to about 4:1 is preferably added.
These processes preferably employ water-soluble enzymes capable of catalyzing stereoselective hydrolysis. Especially suitable for use with these processes are lipases and esterases,:as well as pancreatin and xcex1-chymotrypsin. Either the crude or purified forms of these enzymes, in free form or immobilized on a support (for example, on a resin such as XAD-7, XAD-2 or Accurel(copyright) resins), may be employed. Particularly preferred in these processes are Lipase PS-30 from Pseudomonas sp. (Pseudomonas cepacia) (Amano Int""l), Lipase P-30 (Amano) from Pseudomonas sp., Lipase GC-20 Geotrichum candidum (Amano Int""l), Lipase N Rhizopus niveus (Amano Int""l), Lipase APF Aspergillus niger (Amano Int""l), Lipase AY-30 Candida sp. (Amano), Lipase AK Pseudomonas sp. (Amano Int""l), Pseudomonas fluorescens Lipase (Biocatalyst Ltd.), Esterase 30,000 (Gist-Brocarde), Lipase OF (Sepracor), KID Lipase (Gist-Brocarde), Lipase R (Rhizopus sp., Amano Int.) and Porcine Pancreatic Lipase (Sigma Chem).
The above hydrolyses are preferably conducted in an aqueous, such as a buffered aqueous (e.g., phosphate buffer), medium or in an aqueous medium containing a miscible or immiscible organic solvent. For example, the reaction may be conducted in a biphasic solvent system comprising an organic phase, immiscible in water, and an aqueous phase. Use of a two phase solvent system may enhance the efficiency of such processes where the substrate material is insoluble in water.
Solvents for the organic phase of a biphasic solvent system may be any organic solvent immiscible in water, such as toluene, cyclohexane, xylene, trichlorotrifluoroethane and the like. The aqueous phase is conveniently of water, preferably deionized water, or a suitable aqueous buffer solution, especially a phosphate buffer solution. The biphasic solvent system preferably comprises between about 10 to 90 percent by volume of organic phase and between about 90 to 10 percent by volume of aqueous phase.
An amount of enantiomeric mixture starting material of from about 0.1 to about 100 mg per ml of reaction solution, and one or more enzymes in an amount of from about 0.1 to about 100 mg enzyme per mg of starting material to be hydrolyzed, is preferred.
The reaction mixture is preferably adjusted to and maintained at about pH 7.0, preferably with an aqueous alkali metal hydroxide, carbonate or bicarbonate.
The reaction time may be selected based on the enzyme, the temperature and the enzyme concentration. Temperatures of from about 4xc2x0 C. to about 60xc2x0 C. are preferably employed. 
As can be seen from Reaction Scheme IV above, mixtures I and IV may be selectively dehalogenated to form mixtures II and V, respectively, wherein X denotes a halogen atom.
Any compound capable of effecting these reactions may be employed as the hydroxide ion donor. Exemplary such compounds are selected from water, alkali or alkaline earth metal hydroxides such as sodium and potassium hydroxide, and ammonium hydroxides such as quaternary ammonium hydroxides, for example, those of the formula (R9)4NOH where R9 is hydrogen or alkyl, particularly potassium hydroxide and water. Amounts of the hydroxide ion donor added are preferably those providing a molar ratio of hydroxide ion donor: mixture I or IV enantiomeric starting material of from about 1:1 to about 4:1.
A reaction medium containing water and an organic solvent such as toluene or hexane is preferably employed. The enantiomeric starting materials are preferably employed in an amount of from about 1 mg to about 100 mg per ml of solvent.
Enzymes or microorganisms employed in the dehalogenation reaction are preferably selected from the genera Pseudomonas, Trichoderma, Acinetobacter, Alcaligenes, Nocardia, Mycobacterium, Rhodococcus, Methanobacterium, Proteus, or enzymes derived therefrom, and are preferably employed in amounts of from about 0.1 mg to about 10 mg enzyme per mg of starting material to be dehalogenated.
Temperatures of from about 4xc2x0 C. to about 50xc2x0 C. are preferably employed.
The products of the stereoselective conversions may be isolated and purified by methodologies such as extraction, distillation, crystallization, column chromatography, and the like.
A preferred method for separating the product mixtures formed by the methods of the present invention is by liquid-liquid extraction.
Taxanes are diterpene compounds containing the taxane carbon skeleton: 
which skeleton may contain ethylenic unsaturation in the ring system thereof. Of particular interest are taxanes having the above carbon skeleton wherein the 11,12-positions are bonded through an ethylenic linkage, and the 13-position contains a side chain. Pharmacologically active taxanes, such as taxol analogues, may be used as antitumor agents to treat patients suffering from cancers such as ovarian cancer, melanoma, breast, colon or lung cancer, and leukemia.
The resolved compounds obtained by the methods of the present invention are particularly useful as intermediates in forming the aforementioned C-13 side chain on the taxane skeleton. The addition of such a side chain, in and of itself, may impart an increased or more desirable pharmacological activity to the taxane product, or may form a taxane product which is more readily converted to a taxane having an increased or more desirable pharmacological activity than the starting compound.
The compounds resolved according to the methods of the present invention may be modified prior to use in side chain formation. For example, resolved compounds containing an azide group N3 as the group W may be treated by a reducing agent to form an amine group which may be substituted.
Exemplary methods for side chain formation, and taxane products which may be formed employing such methods, include those described in European Patent Application No. 534,708; U.S. patent application Ser. No. 08/080,704, filed Jun. 28, 1993; U.S. patent application Ser. No. 08/029,819, filed Mar. 11, 1993; U.S. patent application Ser. No. 07/996,455, filed Dec. 24, 1992; U.S. patent application Ser. No. 08/062,687, filed May 20, 1993; U.S. patent application Ser. No. 07/981,151, filed Nov. 24, 1992; and U.S. patent application Ser. No. 07/995,443, filed Dec. 23, 1992; all of which aforementioned documents are incorporated herein by reference.
Salts or solvates of reactants or products may be employed or prepared as appropriate or desired in the methods of the present invention.
The methods of the present invention are further described by the following examples. These examples are illustrative only, and are in no way intended to limit the scope of the instant claims.